Nanoparticles or nanoparticle-based formulations offer the advantage of efficient delivery to the target tissue for enhanced therapeutic or diagnostic purposes, which is usually related to their size, shape, surface properties, and aggregation/agglomeration states. Comprehensive physicochemical characterization of nanoparticles with respect to their size distribution, aggregation/agglomeration state, and shape in aqueous or physiological environments is important, yet challenging, for their use in biomedical applications and compliance with safety regulations. The aggregation/agglomeration of nanoparticles in biological fluids plays a critical role in determining the physical size, shape, and surface properties that are crucial for biological recognition, yet an image-based observation of such aggregation/agglomeration is difficult to achieve in a solid phase. Transmission electron microscope (TEM) is a unique and powerful tool for observing nanoparticles. However, due to the uneven spatial distribution of the nanoparticles in conventional TEM specimens, which are either prepared by drying the solution of nanoparticles onto copper grids or by freezing it on a cryostage holder, the TEM is rarely employed for evaluations of spatial distribution of nanoparticles in aqueous solutions.